Blood, plasma, and biological fluid donation programs are essential first steps in the manufacture of pharmaceutical and blood products that improve the quality of life and that are used to save lives in a variety of traumatic situations. Such products are used for the treatment of immunologic disorders, for the treatment of hemophilia, and are also used in maintaining and restoring blood volume during surgical procedures and other treatment protocols. The therapeutic uses of blood, plasma, and biological fluids require that donations of these materials be as free as possible from viral contamination. Typically, a serology test sample from each individual blood, plasma, or other fluid donation is tested for various antibodies which are elicited in response to specific viruses; particularly hepatitis C (HCV) and two forms of the human immunodeficiency virus (HIV-1 and HIV-2). In addition, the serology test sample may be tested for antigens designated for specific viruses such as hepatitis B (HBV), as well as antibodies elicited in response to such viruses. If the sample is serology positive for the presence of either specific antibodies or antigens, the donation is excluded from further use.
Whereas as an antigen test for certain viruses, such as hepatitis B, is thought to be closely correlated with infectivity, antibody tests may not be so closely correlated. It has long been known that a blood plasma donor may, in fact, be infected with a virus while testing serology negative for antibodies related to that virus. For example, a window exists between the time that a donor may become infected with a virus and the appearance of antibodies elicited in response to that virus in the donor's system. The time period between the first occurrence of a virus in the blood and the presence of detectable antibodies elicited in response to that virus is known as the "window period". In the case of HIV, the average window period is approximately 22 days, while for HCV, the average window period has been estimated at approximately 98 days. Therefore, tests directed to the detection of antibodies may give a false indication for an infected donor if performed during the window period, i.e., the period between viral infection and the production of antibodies. Moreover, even though conventional serology testing for HBV includes tests for both antibodies and antigens, testing by more sensitive methods have confirmed the presence of the HBV virus in samples which were negative in the HBV antigen test.
In order to minimize the possibility of incipient viral contamination of blood, plasma or biological fluid donations which have passed available antibody and antigen tests, the donations are preferably tested by a polymerase chain reaction (PCR) method. PCR is a highly sensitive method for detecting the presence of specific DNA or RNA sequences related to a virus of interest in a biological material by amplifying the viral genome. Because the PCR test is directed to detecting the presence of an essential component of the virus itself, its presence in a donor may be detected almost immediately after infection. There is, theoretically therefore, no window period during which a test may give a false indication of freedom of infectivity. A suitable description of the methodology and practical application of PCR testing is contained in U.S. Pat. No. 5,176,995, the disclosure of which is expressly incorporated herein by reference.
PCR testing is, however, very expensive and since the general donor population includes a relatively small number of donors infected with the viruses of interest, individual testing of each donation is not cost effective or economically feasible. Hence, an efficient and cost-effective method of testing large numbers of blood or plasma donations to eliminate units having a viral concentration above a pre-determined level is required.
One method of testing a large number of plasma donations is to take samples of a number of individual plasma donations and form them into a pool. The pool is then PCR tested and the individual donations comprising the pool are either retained or disposed of depending on the outcome of the PCR test. While reducing the number of PCR tests, and the costs associated therewith, this method results in a substantial waste of a significant number of virus free donations. Since only a single donation with a viral concentration above a pre-determined level will cause a pool to test PCR positive, the remaining donations that contribute to a pool may well be individually PCR negative. This result is highly probable given that a relatively small number of PCR positive donors exist in the general donor population. In the conventional pooling approach, all donations comprising the pool are disposed of upon a PCR positive result, including those donations that are individually PCR negative.
In addition, plasma donations are often frozen soon after they are received. When samples of individual plasma donations are needed for pooling, each donation must be thawed, an aliquot of the blood or plasma removed from the donation, and the donation must then be refrozen for preservation. Multiple freeze-thaw cycles may adversely effect the recovery of the RNA or DNA of interest as well as the proteins contained within the plasma, thus adversely effecting the integrity of the PCR test. Moreover, each time an aliquot of individual plasma donations is withdrawn to form a pool, the donation is subject to contamination, both from the surrounding environment and from the apparatus used to withdraw the aliquot. Further, if the donation contains a virus, it can contaminate other donations. In order to avoid introducing viral contaminants into an otherwise viral free donation, the sample taking apparatus must be either sterilized after each individual use or used for taking only a single aliquot from a single individual donation. A fresh, or sterilized, sample taking apparatus must be used for taking an aliquot from a subsequent individual donation. Either of these methods involves considerable expense and is quite time consuming.
Recently, several advances have been disclosed in the prior art that relate to systems and methods for preparing pools of individual plasma donation samples for PCR testing. In particular, U.S. Pat. No. 5,591,573 describes a cost-effective and efficient process for preparing and testing samples from a multiplicity of blood or plasma donations to uniquely identify donations which are infected with the virus as well as systems and devices for practicing the process. A flexible collection segment is connected to a blood or plasma donation collection container and is in fluid communication with the inside of the container. The collection segment is filled with blood or plasma from the container and a portion of the collection segment is heat sealed at both ends. The sealed portion of the collection segment is removed from the container and, either before or after the sealed collection segment portion is removed, spaced-apart heat seals are provided at regular intervals along the length of the segment between the sealed ends. The segment portions in the intervals between adjacent seals define sample pouches which each contains a plasma or blood sample. The tubing segment, which has been converted into a series of pouches, has been disconnected from the plasma collection bottle and frozen until needed for testing.
To begin the testing process, a first pouch is removed from each of a group of segments and a portion of the contents of each first pouch is withdrawn and formed into a pool. Depending on the test results of an initial pool, aliquots may be taken from additional sample pouches of each of the plasma donations and formed into subpools. The process is iterated, with each viral positive pool being further subdivided into successively smaller subgroups, with each of the successive subgroups comprising a fraction of the samples of the preceding positive subgroup, until the final pouch corresponding to a single viral positive plasma donation is identified.
In order to form a generational pool, appropriate sample pouches, having the same generational index as the pool to be formed, are arranged on a titer plate and held in place by a cover which contains access openings though which sample pouches may be accessed by a cannula. A portion of the contents of each sample pouch is withdrawn and the contents formed into a pool in a pooling container.
While relatively simple and cost effective, such a system of manual sample extraction is time consuming and requires a laboratory clinician to pay careful attention to each step of the process. In particular, care must be taken in order to prevent carry-over contamination, i.e., a clean or sterilized cannula must be used to withdraw samples for each generational pool. Accordingly, with a manual sample extraction system, used cannulas which may be contaminated with HBV, HIV-1 or HIV-2 virus, must be removed from the sample extraction apparatus for either sterilization or replacement. The dangers attendant with manual manipulation of "sharps" of this type are significant and well known.
Accordingly, there exists a need for an automated system and method for preparing pools and subpools for PCR testing, which enhances safety by minimizing the degree of manual intervention and manipulation. Such a system and method should be able to extract aliquots from blood and/or plasma donation samples in a manner which minimizes the potential for environmental contamination through "splashing" or "out-gassing", while also minimizing the volume of potentially hazardous materials which must be disposed of.